Defining the Substrate Specificity Determinants Recognized by the Active Site of C-Terminal Src Kinase-Homologous Kinase (CHK) and Identification of β-Synuclein as a Potential CHK Physiological Substrate

C-Terminal Src kinase-homologous kinase (CHK) exerts its tumor suppressor function by phosphorylating the C-terminal regulatory tyrosine of the Src-family kinases (SFKs). The phosphorylation suppresses their activity and oncogenic action. In addition to phosphorylating SFKs, CHK also performs non-SFK-related functions by phosphorylating other cellular protein substrates. To define these non-SFK-related functions of CHK, we used the kinase substrate tracking and elucidation method to search for its potential physiological substrates in rat brain cytosol. Our search revealed beta-synuclein as a potential CHK substrate, and Y127 in beta-synuclein as the preferential phosphorylation site. Using peptides derived from beta-synuclein and positional scanning combinatorial peptide library screening we defined the optimal substrate phosphorylation sequence recognized by the CHK active site to be E-x-[Phi/E/D]-Y-Phi-x-Phi, where Phi) and x represent hydrophobic residues and any residue, respectively. Besides beta-synudein, cellular proteins containing motifs resembling this sequence are potential CHK substrates. Intriguingly, the CHK-optimal substrate phosphorylation sequence bears little resemblance to the C-terminal tail, sequence of SFKs, indicating that interactions between the CHK( active site and the local determinants near the C-terminal regulatory tyrosine of SFKs play only a minor role in governing specific phosphorylation of SFKs by CHK Our results imply that recognition of SFK, by CHK is mainly governed by interactions between motifs located distally from the active Site of CHK and determinants spatially separate from the C-terminal regulatory tyrosine in SFKs. Thus, besides assisting in the identification of potential CHK physiological substrates, our findings Shed new light on how CHK recognizes SFKs and other protein substrates.,